The determination of calcium in biological fluids using an orthocresolphthalein complexone reagent composition is an accepted method in many hospital, industrial, reference and commercial clinical laboratories. The method is based on the use of the acid-base indicator orthocresolphthalein complexone, also named as 3',3"-bis([bis(caboxymethyl)-amino] -methyl)-5',5"-dimethylphenolphthalein and abbreviated as "OCPC", to form a measurable color with calcium under alkaline conditions. A neutral or acid solution of the compound is colorless; alkaline solutions are violet, the intensity of the color deepening as the pH increases.
The method is usually carried out by mixing a color reagent, contaning orthocresolphthalein complexone in a dilute hydrochloric acid solution, and an alkaline reagent, containing an amine base and a small amount of an alkali metal cyanide, with a biological fluid. The acid and the amine base provide an alkaline buffer which gives a suitable pH, usually pH 11, in the ultimate mixture. The intensity of the resulting color is measured photometrically after a predetermined time interval at a predetermined temperature. orthocresolphthalein complexone methods for determination of calcium have been described, for example, by Stern et al., Clin. Chem. 2, 576 (1957); Gitelman, Analytical Biochemistry, 18, 521 (1967) and Connerty et al., Am. J. Clin. Path. 45, 290 (1966). The determination may be carried out in the presence of a small amount of 8-quinolinol, to minimize interference by magnesium ion, as taught by Connerty et al., supra. Potassium cyanide has also been employed in such methods to minimize interference by heavy metals.
Diethylamine is generally employed as the amine base. Diethylamine has an offensive odor and a low flash point, and diethylamine reagents are unduly sensitive to both pH and calcium concentration. It would be desirable to eliminate its use in calcium determinations. Connerty and Briggs, supra, described a reagent in which a 0.74 molar, pH 11 aminoethanol borate buffer (AEB) is employed. The AEB system required protein precipitation or calcium oxalate precipitation before analysis, and is thus unsuitable for direct analyses on small serum samples. Also, the AEB reagent is oversensitive to calcium, giving absorbance values of 0.5 and higher for calcium levels near the high end of the normal range, making the results difficult to read accurately.